WO2003080837A1

WO2003080837A1 - Production of recombinant che a 1 allergen from chenopodium album and system of isolating and purifying same

Info

Publication number: WO2003080837A1
Application number: PCT/ES2003/000135
Authority: WO
Inventors: Rosalia Rodriguez Garcia; Maria Teresa Villalba Diaz
Original assignee: Universidad Complutense De Madrid Rectorado
Priority date: 2002-03-22
Filing date: 2003-03-21
Publication date: 2003-10-02

Abstract

The invention relates to the production of the Che a 1 protein, using recombinant technology, in Pichia pastoris yeast which involves the use of nucleotide sequence SEQ ID NO:1 or other nucleotide sequences obtained by mutagenesis of sequence SEQ ID NO:1. For this purpose, a main characterised and purified allergen is isolated from Chenopodium album pollen, said allergen being recognised by more than 60 % of patients allergic to said biological source. In this way, the recombinant DNA which codes for the Che a 1 allergen from goosefoot pollen (Chenopodium album) has been cloned and expressed. Said allergen shows homology with ryegrass and birch proteins. The recombinant protein produced in yeast (Pichia pastoris) is isolated with high yields, is correctly folded and exhibits immunological properties equivalent to those of the natural allergen. Hence, said protein and the homologous proteins thereof, the modified forms derived from same and derived fragments containing at least one allergen epitope can be used in immunotherapy and diagnosis protocols.

Description

Production of the recombinant Chenopodium allergen. Che a l album, and isolation and purification system.

OBJECT OF THE INVENTION

The present invention, as expressed in the statement of this specification, refers to one of the allergens of the flea pollen {Chenopodium album), the Che a l protein, to allergenic epitopes present in the protein. The invention also makes reference to the recoding DNAs that encode both complete proteins and fragments that include at least one epitope of the molecule, as well as homologous molecules of Che a 1 with allergenic capacity in other related and unrelated species. Specifically, the object of the invention consists in the production of the Che a 1 protein, by recombinant technology in the yeast Pichia pastoris, which implies the use of the nucleotide sequence characterized by SEQ ID NO: 1 or other nucleotide sequences obtained by mutagenesis of the sequence SEQ ID NO: 1.

The invention also provides an effective method of isolation for both the recombinant and the natural allergen.

STATE OF THE TECHNIQUE

Type I allergy is a disease that affects more than 20% of the population of industrialized countries. This condition is caused by allergens, present in both organisms and biological products - food, mites, insect poisons, pollens, fungi, mammalian epithelia - and in synthetic materials. In most of the allergenic biological sources, the allergens they are proteins of molecular masses between 5 and 70 Da. Symptoms that derive from allergy, such as rhinitis, conjunctitis, or asthma, are caused by the release of cellular mediators, such as histamine, from basophils and astocytes, immune system cells. Said release is induced by cross-linking of IgE antibodies bound to high affinity receptors.

FcεRI, which are in turn anchored to basophils and mast cells. The cross-linking of the IgE is caused by the binding of the corresponding allergen, or a fragment thereof, through an IgE epitope (contained in said allergen, or in its fragment). The therapy that is currently being used to treat the allergy involves the hyposensitization of the patient by parental or oral administration of adequate doses of the allergen itself, or related allergoids. In almost all cases, an allergenic extract obtained by means of a minimal manipulation of the natural biological source is administered, which implies a very complex mixture of proteins and other substances, in which the allergen - or allergens - can represent a very small part of the total product used.

In fact, at present, the protocols used for the diagnosis of allergy cases and their subsequent immunotherapy involve the use of allergenic extracts that are often not characterized, or even standardized with respect to the most important allergens they may contain. Often, its administration does not provide a complete diagnosis, especially when the patient's hypersensitivity refers to allergens present in low concentration in said extracts. On the other hand, immunotherapy performed with these preparations is often ineffective and sometimes causes undesirable side effects that may lead to to be more serious than the allergic condition that is intended to resolve. An alternative to the use of these extracts is the preparation of mixtures of the most significant allergens, obtained by isolation from their natural source. However, this route presents two important barriers. On the one hand, it implies a good knowledge of the allergenic components of the material that causes the allergy, at least of all its main allergens, and this data is frequently not available. On the other hand, the process of isolation of known allergens, from biological material, is often difficult and does not provide the necessary quantities or the quality required for subsequent use, mainly due to the low levels in which it is found. All these problems are accentuated when the allergenic material is the pollen of a plant species. The large amount of pigments, carbohydrates, lipids and insoluble components make handling very difficult. It goes without saying that its availability is also very low and of high economic cost, due to the difficulty of its collection. For all the above, it is necessary to design procedures for the production of allergens to be used in allergy diagnosis and immunotherapy protocols. The production of recombinant DNA is predicted as the most reproducible and effective way to obtain well-defined allergenic polypeptides, not only for clinical use, but even for research.

The polinosis to frown is acquiring a certain relevance as responsible in the induction of allergy in countries of the Mediterranean area and desert areas, where this bush is widely extended (Galán C. et al (1989) Ann. Allergy 63, 435-437; Subiza J. et al (1998) Rev. Esp. Alergol. Immunol. Clin. 13, 45-58; Suli an FA et al (1997) Ann. Allergy Asthma Immunol. 78, 415-418). In the At present, little is known about the allergenic molecules in this biological source, this allergen being the first to be characterized.

EXPLANATION OF THE INVENTION

Production of the Chenopodi recombinant allergen um album Che a 1 in the yeast Pichia pastoris, and isolation and purification system. The present invention relates to the method of isolation used for the purification of the Che allergen to 1 - an allergen of the frown pollen of increasing clinical importance, with a prevalence of 60% of the patients allergic to the pollen of the frown - from the pollen of Frown (Chenopodium album). More specifically it comprises obtaining, by cloning, DNA molecules. recombinant encoding polypeptides with the same or similar antigenic properties as the allergen from Chenopodium pollen, or polypeptides that have at least one epitope, antigenic or allergenic, of the allergen Che a l. The invention also includes the complete sequence of the cDNA of the Che 1 allergen and the complete sequence deduced from the allergen.

The process object of the invention allows the purification of Che 1 allergen of the pollen from the pollen from a pollen protein extract by means of a penetrability chromatography and HPLC, as described in detail below.

In this way, recombinant DNA molecules that encode polypeptides that exhibit the antigenicity of Che to 1 are obtained, as well as polypeptides that contain at least one epitope of Che to 1 in their structure. The invention has polynucleotide DNA sequences that hybridize, under conditions restrictive, with those described above - which implies a level of identity of at least 60% between their nucleotide sequences - or are derived from them by degeneracy of the genetic code or mutagenesis. The cloning procedure of the Chenopodium album Che a 1 recombinant allergen includes cloning vectors and host cells that contain a nucleotide sequence as described in SEQ ID NO: 1, Che 1 coding, homologous proteins or fragments thereof.

Therefore, nucleotide fragments are obtained that have at least one sequence that encodes an antigenic determinant of Che 1 allergen or of fragments thereof, as well as homologous allergens of Che 1, mainly in species related to the brow that , given the structural similarity, they present allergenic cross-reactivity with Che alo with a part of it. Among the species related to the frown are: Betula verrucosa (birch) and Lolium perennial (ballico) with a similarity of 70% and 51%, respectively.

These polypeptides may contain the antigenic sequence linked to other polypeptides (for example as fusion proteins), have been chemically synthesized or have been obtained by proteolytic digestions and chemically or enzymatically modified.

The insertion of said sequence into expression vectors in eukaryotic host organisms makes it possible to have recombinant constructs that can be used for the production of the recombinant allergen. Once the polypeptide molecule with antigenic activity of Che alo epitopes B and T of its own is produced, it is isolated from the rich extracellular medium of the culture in soluble form by anion exchange chromatography, penetrability and HPLC or from the minimum medium by penetrability chromatography and HPLC.

The recombinant products obtained by the method object of the invention have the ability to bind IgE antibodies from serum of patients allergic to gum, sensitive to Che a 1.

Finally, with the process object of the invention, Che 1 is available immunologically active, in addition to antigenic and allergenic fragments of this allergen, and homologous proteins thereof, which serve to be incorporated into the "in vivo" and "tests" in vitro "to be carried out for the faithful diagnosis of hypersensitivity to this pollen, and to other pollen related phylogenetically with it. They may also be used in allergen preparations that are used to carry out the corresponding immunotherapy for the treatment of allergy to pollen of the eyebrow.

Protein extracts obtained from pollen are currently used for the diagnosis and therapy of flea allergy. This implies low reproducibility and a high content of contaminating molecules, of protein and non-protein origin, which can cause adverse side effects in treated patients. The availability of homogeneous molecules obtained by recombinant DNA techniques, in unlimited quantities, perfectly quantifiable and standardized, considerably reduce all the aforementioned inconveniences. This technology makes it possible to have these molecules and, in addition, peptides or modified forms thereof containing at least one of the allergenic epitopes.

EMBODIMENT OF THE INVENTION

The present invention relates to a recombinant DNA encoding epitopes of Chenopodium album allergen Che a 1, to recombinant production, in Pichia yeast pastoris, of the polypeptides encoded by said DNA and to the isolation of the natural allergen from the pollen of the brow. It also refers to the application of both molecules in the diagnosis and therapy of this immune disorder.

In the process of cloning the Chenopodium recombinant allergen album Che a 1, the N-terminal sequence was obtained by Edman degradation. Part of that sequence (Cys Arg Val Gln Pro Met Thr) served to design the degenerate oligonucleotide, NCH3: 5'-TGYCGNGTNCARTTYATGAC-3 that was used to amplify by PCR the corresponding coding (16-143) and non-coding 3 'corresponding sequence. to Che cDNA 1. These sequences subsequently allowed to obtain the C-terminal sequence, (Asp Leu Tyr Asp Val Lys Ala Asn), with which the non-degenerated oligonucleotide NCH4 was designed: cgtccgcggttaATTAGCTTTAACATCATAAAGATC. Cloning of the amplification product with the non-degenerated oligonucleotides, NCH5: cgtctcgagaaaagaGCCGAGAACCATTTCAAAGTC

(Ala Glu Asn His Phe Lys Val) and NCH4 allowed to obtain the nucleotide sequence coding for the complete mature protein, a sequence that was used to carry out the expression. The procedure object of this invention is carried out by the following phases:

• Total RNA isolation

Total RNA was isolated from brow pollen following the protocol published by Ullrich et al. [Science 196, 1313-1318, 1977]. In this isolation, 0.5 g of pollen was started and homogenized in a 0.1 M Tris-HCl buffer, pH 7.5 containing 4 M guanidinium thiocyanate, 0.5% sodium lauryl sarcosinate and 2- 0.14 M mercaptoethanol, using a Polytron homogenizer. This suspension was centrifuged at 5000 xg at 20 ° C in a Sorvall centrifuge. The supernatant was subsequently subjected to a gradient centrifugation of 5.7 M CsCl in 0.01 M EDTA at 40,000 rpm in an S 60 rotor (Beckman) for 12 hours. From 5 μg of total RNA cDNA synthesis was carried out using the ^Λ "SMART II cDNA synthesis kit (Clontech).

• Cloning of Che to 1 based on PCR techniques

As already mentioned above, the primers corresponding to the N- and C-terminal sequences used were designed taking into account the protein sequence obtained by Edman degradation and by sequencing of the PCR fragment, respectively. The method used was based on a multi-stage PCR reaction. In the first one, the complete Che to 1 sequence is obtained, including the 3 'non-coding sequence of this allergen. For this, the Che-1 specific cDNA is used as a template and as primers, the degenerated oligonucleotide NCH3, corresponding to a sequence contained in the N-terminal sequence of the protein determined by Edman degradation, and the unspecific oligonucleotide UPM: 5 ' - CTAATACGACTCACTATAGGGC-3 '. The mold and the primers NCH3 and

UPM were dissolved in a standard PCR mixture (250 μM of dNTPs, standard buffer and 50 pmoles of the primers). After a first stage of denaturation at 95 ° C for 15 min, 25-35 cycles were carried out at hybridization temperatures of 42-55 ° C in the presence of Advantage DNA polymerase II (Clontech). The reaction was electrophoresed in 1.5% agarose gels and the DNA fragments were purified using the Magic PCR Prep kit (Promega) and inserted into the linearized plasmid pCR2.1 and with a terminal T at the 5 'ends. With this construction, TOP10 competent cells were transformed, then the recombinants were selected and the sequencing of some of the selected clones was carried out, obtaining sequences of this allergen. In a second PCR stage using the NCH4 and UPM oligonucleotides the coding sequence has been determined including that corresponding to the protein signal peptide. In the last stage of PCR, and using the non-degenerated oligonucleotides NCH4 and NCH5, the DNA fragments containing the coding sequence corresponding to the mature allergen were obtained.

For the production of the recombinant form of this protein, the DNA coding sequence was linked to the expression plasmid pPICZoA (Invitrogen Corp.). The cells used were from the KM71 strain of Pichia pastorís. Induction was carried out by 0.5% methanol for five days, the cells growing at 30 ° C in rich medium or minimal yeast growth medium. The cells were pelleted by centrifuging at 5000 xg, the supernatants were collected and a sample thereof was applied on a 15% polyacrylamide gel in the presence of SDS. A unique 19.5 kDa band was detected by staining the gels with Coomassie Blue. The protein is recognized by sera from specific allergic patients from Che to 1 natural (1:10 dilution). The purification of the proteins obtained in minimal medium was carried out by lyophilization of the extracellular medium of the yeast and the application of two chromatographic stages, one of penetrability in Sephadex G-75 and another in a C-18 reverse phase column in HPLC, obtaining after the latter the allergen with a degree of purity of 99%. For the production of the allergen in rich medium a stage was carried out additional ion exchange in DEAE-cellulose with the dialyzed extracellular medium and then the two chromatographic stages, penetrability and HPLC, were carried out with the protein produced in minimal medium. Structural characterization studies (far UV spectra, mass spectrometry) and immunology (with individual sera of allergic patients) were carried out with them, and in all cases the recombinant proteins behaved in a manner equivalent to that of the natural allergen.

• Isolation of the Che a l allergen from the pollen of the brow (Chenopodium album).

Che 1 allergen has been purified from protein extract from. Brow pollen, obtained by ho ogenization of 3g of pollen in 50 mM ammonium bicarbonate pH 8.0. Said extract was applied to a column of Sephadex G-75. Fractions containing the protein were applied to a C-18 reverse phase column in HPLC with a gradient of 0-60% acetonitrile in 0.1% TEA.

The invention comprises the use of recombinant Che a 1 in diagnosis, "in vivo" by means of skin tests or "in vitro" by immunodetection techniques (ELISA, RAST), allowing to specify which allergens are responsible for the clinical symptoms of an individual and reduce the number of molecules necessary for your immunotherapy. Thus, a personalized immunotherapy is originated.

Claims

1. Recombinant DNA molecules characterized by SEQ ID NO: 1 that encode polypeptides with allergenic activity and that possess at least one common allergenic epitope with the Che a 1 allergen from Chenopodium album.

2. Recombinant DNA molecules, or modifications thereof, according to claim 1, which encode polypeptides with at least one Che to 1 epitope in their structure, attached to an additional polypeptide, or chemically or enzymatically modified.

3. Recombinant DNA molecules characterized by hybridizing under strongly restrictive conditions with that described in SEQ ID NO 1.

4. Recombinant DNA molecules characterized by presenting at least 60% identity with that described in SEQ ID NO: 1, containing a nucleotide sequence encoding a polypeptide with identical or reduced allergenic activity (hypoallergenic) with respect to Che to 1.

5. Recombinant polypeptide corresponding to Che a 1 allergen from Chenopodium album, or fragments of said polypeptide, according to claims 1 and 2, characterized by the sequence given in SEQ ID NO: 2 and presenting the antigenicity of at least one epitope of Che to 1.

6. Recombinant polypeptides of the homologous allergen of Che to 1 in other related and non-phylogenetically related species or fragments of said polypeptides, according to claims 3 and 4 that present the antigenicity of at least one epitope of Che to 1.

7. Polypeptides having an amino acid sequence according to claims 5 and 6, and having an attenuated allergenicity.

8. Polypeptides or fragments of said Chenopodiaceous pollen polypeptides with at least one Che to 1 epitope, according to previous claims, that recombinants bound to an additional polypeptide are produced.

9. Pollen polypeptides of Chenopodium album, according to previous claims, which are chemically or enzymatically modified.

10. A eukaryotic expression vector, and preferably pPICZαA, containing the sequences characterized by SEQ ID NO: 1, according to claims 1,2,3 and 4, whose expression results in the production of the polypeptides characterized by SEQ ID NO: 1 and 2 according to claims 5,6,7,8 and 9.

11. A eukaryotic host organism, preferably Pichia pastoris yeast, transformed with the construction formed according to claim 10.

12. A method for producing the recombinant forms corresponding to the polypeptide sequences described in claims 5,6,7,8 and 9, in the eukaryotic system of yeast Pichia pastoris, and preferably of strain KM71, grown in rich medium, characterized by an ion exchange chromatography in DEAE-cellulose, one of penetrability in Sephadex G-75 and one in a C-18 reverse phase HPLC column.

13. A method for producing the recombinant forms corresponding to the polypeptide sequences mentioned in claims 5,6,7,8 and 9, in the eukaryotic system of yeast Pichia pastoris and preferably of strain KM71 grown in minimal medium, characterized by a Sephadex G-75 penetrability chromatography and one on a C-18 reverse phase HPLC column.

14. A method for the isolation of the Che a l allergen from the album Chenopodium pollen using a penetrability chromatography in Sephadex G-75 and another in HPLC reverse phase C-18 nucleosyl.

15. Use of the polypeptides of claims 5-9, as pharmacological molecules

16. Use of the molecules, according to claims 1-9, in the in vitro ^λ diagnosis of allergy.

17. Application of the recombinant molecules of claims 1-9 for the design of vaccines intended for allergy immunotherapy.

18. Application of the recombinant molecules of claims 1-4 to produce peptides, according to claims 5-9, containing at least one allergenic T epitope, capable of acting as vaccines.

19. Application of the recombinant molecules of claims 1-4 in the production of hypoallergenic recombinant isoforms that have decreased or nullified their binding to IgE for the treatment of allergies.